In biology it is of interest to mark structures such as cells or viruses with fluorescent materials for accurate identification, ease of detection and microscopic analysis. Traditionally, organic dye fluorophores have been the favored materials and have the capability to be modified with a range of materials, enabling targeted binding to a wide range of biological structures based on known affinities and chemistries. Upon binding of the dye to the target biological material, an activating light of a given wavelength is used to excite the dye, from which it responds by fluorescently emitting a characteristic light radiation specific to the properties of the organic dye employed. However, traditional organic dyes have numerous limitations when used to tag biological materials.
Semiconductor fluorescent nanocrystals (“quantum dots”) are nanometer sized semiconductor, light-emitting crystals, spherical in shape and have superior fluorescent properties to organic dyes. Quantum dots are generally synthesized with Type II-VI (e.g. CdSe, CdTe, CdS and ZnSe) or Type III-V (e.g. InP and InAs) column elements from the periodic table and can be capped with numerous shells, layers or molecules to modify their physical properties, such as for surface functionalization. Integration of quantum dots in biology was achieved in breakthroughs showing that highly luminescent quantum dots could be made water-soluble and subsequently biocompatible using surface modification techniques such as silica/siloxane coatings or direct absorbtion of bifunctional ligands, which presented them useful tools in biology. Quantum dots are emerging as the new biological label with applications and properties superior to traditional fluorescent proteins and organic dyes.
Most of the limitations with traditional organic dyes are a result of the extremely limited absorptive and emissive capabilities. The first shortcoming is that the peak emission of organic dyes cannot be altered—each dye corresponds to a different molecule with a different pre-set emission wavelength, or fluorescent color, that is set by nature. The second shortcoming is the narrow absorption pattern of organic dyes—dyes tend to display absorption peaks that are not always in convenient regions of the spectrum, making the excitation of various organic dyes challenging and costly. The third shortcoming is that of uneven absorption and emission peaks—organic dyes have a tendency to produce “shoulders” in the geometry of their emission and absorption peaks, which is a major disadvantage in applications that require Gaussian type emission patterns to work correctly. An additional shortcoming is that of stability—the lifetime of organic dyes varies but is generally low relative to that of other tagging mechanisms and organic dye fluorescence is controlled entirely by the molecular bonding properties of each individual dye. Finally, incident radiation absorbed by an organic dye molecule moves electrons into excited states, whereupon they decay and release light radiation. This emission cannot be altered because it corresponds to pre-set excited states of the dye molecule that are inherent to every molecule of that type.
Whereas the light emission ranges and possible forms of organic dyes are very limited, quantum dots can be made to emit light at any wavelength in the visible and infrared ranges, and can be inserted almost anywhere, including in liquid solutions, dyes, paints, epoxies, and sol-gels. Furthermore, quantum dots can be attached to a variety of surface ligands, and inserted into a variety of organisms in vivo or in vitro.
Numerous methods exist for covalently linking biological molecules to quantum dots to create a bio-molecular conjugates (“bioconjugate”) or functional quantum dot which are used in labeling, detection and imaging applications to attach or bind a quantum dot to a biological material based on specific chemical or biological affinity. These methods employ a variety of chemistries to water-soluble quantum dots from which several cross-linker molecules can be coupled to enable the attachment of the primary functional biomaterial. Other examples of bioconjugate techniques enabling the attachment of various materials to quantum dots are known to those skilled in the art.
Generally, bioconjugation methods are classified into mechanisms using: (1) Biofunctional linkages, (2) Electrostatic attraction, (3) Hydrophobic attraction, (4) Silanization, and (5) Nanobead linkages. Examples of methods employing bioconjugative techniques are polyethylglycol modification of the underlying carboxyl quantum dots, and optimization of the surface loading of amino groups for high conjugation efficiency and specificity. Another example is modifying the quantum dots with peptides through the amino or carboxyl groups at the terminus, or using other residues, small molecules, proteins, or nucleic acids, and other methods known to those skilled in the art. More specifically, schemes used for the conjugation of antibodies to quantum dots are based on well-known chemistries using the fast and efficient coupling of thiols to maleimide groups, with reactive groups such as primary amines, alcohols, carboxylic acids and thiols used to link the antibodies to the quantum dots.
Quantum dots represent a marked increase in performance over standard organic dyes, because they can be tuned to absorb or emit at any visible or infrared wavelength and can be fabricated into a great variety of forms and media, eliminating completely the shortcomings of dyes. These unique abilities are due to their very small sizes (typically between 1-10 nm in diameter). The small size and its direct relationship to fluorescence also allows for incredible versatility and flexibility of form, letting phosphors match whatever shape their underlying light-emitting diode (LED) needs to assume.
When light impinges on quantum dots, it encounters discretized energy bands specific to the quantum dot. The discretized nature of quantum dot bands means that the energy separation between the valence and conduction bands (the bandgap) can be altered with the addition or the subtraction of just one atom—making for a size dependent bandgap. Pre-determining the size of the quantum dots fixes the emitted photon wavelength at the appropriate customer-specified color, even if it is not naturally occurring—an ability limited only of quantum dots.
Additionally, it is also known that certain rare-earth metal chelates emit visible light upon irradiation with UV light and different forms of visible light (e.g., violet or blue light), an emission which is characterized by the chelated cation. Some lanthanide ions, such as those of europium (Eu3+), Samarium (Sm3+), terbium (Tb3+), and to a lesser extent dysprosium (Dy3+) and neodymium (Nd3+), exhibit typical fluorescence characterized by the ion, especially when chelated to suitable excitation energy mediating organic ligands. The fluorescent properties of these compounds—long Stokes' shift, narrow band-type emission lines, and unusually long fluorescence lifetimes—have made them attractive candidates for fluorescent immunoassays and time-resolved fluorometric techniques.
The major emission lines of these fluorescent lanthanide chelates are formed from a transition called hypersensitive transition and are around 613-615 nm with Eu3+, 545 (and 490) nm with Tb3+, 590 and 643 nm with Sm3+, and 573 with Dy3+. Radiation is typically absorbed by the chelates at a wavelength characteristic of the organic ligand and emitted as a line spectrum characteristic of the metal ion because of an intramolecular energy transfer from the ligand to the central metal ion. The organic ligand absorbs energy and is raised or excited from its singlet ground state, S0, to any one of the vibrational multiplets of the first singlet excited state, S1, where it rapidly loses its excess vibrational energy. At this point, there are two possibilities: relaxation by an S1→S0 transition (ligand fluorescence) or intersystem crossing to one of the triplet states, T1.
Fluorescent europium chelates are known to exhibit large Stokes shifts (˜290 nm) with no overlap between the excitation and emission spectra and very narrow (10-nm bandwidth) emission spectra at 615 nm. In addition, the long fluorescence lifetimes (measurable in microseconds instead of the nanosecond lifetimes measurable for conventional fluorophores) of the chelates help filter out noise and other interference having a low fluorescent lifetime. The long fluorescent lifetimes thus permit use of the chelates for microsecond time-resolved fluorescence measurements, which further reduce the observed background signals. Additional advantages of using europium chelates include that europium chelates are not quenched by oxygen.
In specific binding assays, sensitivity is of prime importance due to the generally low analyte levels that are measured. Radioimmunoassay sensitivity limits the assay to measurements of concentration of 10−12 M, and more often only in the 10−8 to 10−10 M range. In addition, radiolabels suffer from the drawbacks of short half life and handling hazards.
In fluorescence spectroscopy assays, a sample containing a fluorescent species to be analyzed is irradiated with light of known spectral distribution within the excitation spectrum of the target fluorescent species. The intensity of the resulting characteristic emission spectrum of the fluorescent target molecules is determined and is related to the number of target molecules.
The sensitivity of fluorescence assays, although theoretically very high, is limited by the presence of background fluorescence. Background signal levels are picked up from competing fluorescent substances, not only in the sample, but also in materials containing the sample. This is an especially serious problem in quantitative measurements of species associated with samples containing low concentrations of desired target fluorescent molecules such as found in biological fluids. In many situations, it is impossible to reduce the background sufficiently (by appropriate filtration and other techniques known in the art) to obtain the desired sensitivity.
Time resolution offers an independent means of isolating the specific fluorescent signal of interest from nonspecific background fluorescence. Time resolution is possible if the label has much longer-lived fluorescence than the background, and if the system is illuminated by an intermittent light source such that the long-lived label is measurable during the dark period subsequent to the decay of the short-lived background.
Certain fluorescent molecules have been commonly used as tags for detecting an analyte of interest. Organic fluorescent dyes are typically used in this context. However, there are chemical and physical limitations to the use of such dyes. One of these limitations is the variation of excitation wavelengths of different colored dyes. As a result, the simultaneous use of two or more fluorescent tags with different excitation wavelengths requires multiple excitation light sources.
A drawback of organic dyes is the deterioration of fluorescence intensity upon prolonged and/or repeated exposure to excitation light. This fading, called photobleaching, is dependent on the intensity of the excitation light and the duration of the illumination. In addition, conversion of the dye into a nonfluorescent species is irreversible. Furthermore, the degradation products of dyes are organic compounds which may interfere with the biological processes being examined.
Additionally, spectral overlap exists from one dye to another. This is due, in part, to the relatively wide emission spectra of organic dyes and the overlap of the spectra near the tailing region. Few low molecular weight dyes have a combination of a large Stokes shift, which is defined as the separation of the absorption and emission maxima, and high fluorescence output. In addition, low molecular weight dyes may be impractical for some applications because they do not provide a bright enough fluorescent signal.
Furthermore, the differences in the chemical properties of standard organic fluorescent dyes make multiple, parallel assays impractical as different chemical reactions may be involved for each dye used in the variety of applications of fluorescent labels.
Thus, there is a continuing need in the assay art for labels with the following features: (i) high fluorescent intensity (for detection in small quantities), (ii) adequate separation between the absorption and emission frequencies, (iii) good solubility, (iv) ability to be readily linked to other molecules, (v) stability towards harsh conditions and high temperatures, (vi) a symmetric, nearly gaussian emission lineshape for easy deconvolution of multiple colors, and (vii) compatibility with automated analysis. At present, none of the conventional fluorescent labels satisfies all of these requirements.
While fluorescent emissions from functional quantum dot bioconjugates have been used to detect the presence or absence of a target substrate in a sample, at present there remains no fast and effective method and apparatus for measuring SAM and SAH.